Analysis of adaptive response to DNA damage checkpoint induced arrest

ANALYSIS OF ADAPTIVE RESPONSE TO DNA DAMAGE CHECKPOINT INDUCED ARREST YIO WEE KHENG NATIONAL UNIVERSITY OF SINGAPORE 2009 ANALYSIS OF ADAPTIVE RESPONSE TO DNA DAMAGE CHECKPOINT INDUCED ARREST YIO WEE KHENG B.Eng.(Hons.), M.Sc., NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Acknowledgements Professor Uttam Surana, Dr Lim Hong Hwa, and Dr Dave Wee are the three persons that I can never thank enough for their guidance and assistance. Without their advice and selfless support, this thesis would never have become a reality. Prof Surana is my supervisor. He is truly an excellent scientist; a role model to learn from. Dr Lim is a friend and mentor; an efficient and proficient experimentalist. She taught me most of the molecular biology techniques. Dr Wee is a brilliant budding scientist. He has many marvelous ideas which are still in progress. I would like to express my gratitude to Prof Baltazar Aguda, my ex-supervisor, who has taught me computational modeling of biology. Next, I would also like to thank the members of my Thesis Advisory Committee: Prof Mohan Balasubramanian (TLL) and Prof Wang Yue (IMCB) for their constructive comments. Special thanks to the special people, the current and ex-members of US Lab (IMCB), who have helped me in one way or another. They are San Ling, Zhang Tao, Joan Cher, Jenn Hui, Hong Qing, David Goh, Karen Crasta and Jonathan Wong. I would like to thank Dr Jayantha Gunraratne and Associate Professor Walter Blackstock (IMCB) for the collaboration on mass spectrometry, Prof James Haber (Brandeis Un\iversity) and Prof Rodney Rothstein (Columbia University) for providing valuable reagents. I would also wish to thank the staff at the NUS Graduate School for Integrative Sciences and Engineering (NGS), A*STAR Graduate Academy (AGA) and Institute of Molecular and Cell Biology (IMCB), for their assistance. Most importantly, I wish to thank my family for their support. i Preface After completing my degrees in electrical engineering and bioinformatics, I initiated my PhD studies under the supervision of Professor Baltazar Aguda at the Bioinformatics Institute (A*STAR), Singapore. The research project was purely computational in nature and involved analysis of biological networks. However, after 1.5 years into my PhD program, Professor Aguda decided to move to the USA due to unforeseen circumstances. To continue my PhD program, I joined Professor Uttam Suranas lab at the Institute of Molecular and Cell Biology (A*STAR, Singapore) and undertook the analysis of DNA damage response in budding yeast. The remaining years of my graduate studies were spent first learning the genetic and molecular biological techniques and then analyzing cells adaptation to DNA damage. The format of this thesis reflects this change in the circumstances surrounding my PhD program. The thesis consists of two parts: experimental (Chapters 3, 4) and computational (Chapter 5). The first part (experimental) focuses on investigating the role of Cdc5 polo-like kinase in adaptation, whereas the second part (computational) attempts to unravel the mechanism of gene-expression response due to oscillatory transcription factors. ii Table of Contents ACKNOWLEDGEMENTS I PREFACE II TABLE OF CONTENTS . III SUMMARY . V LIST OF TABLES . VIII LIST OF FIGURES IX LIST OF SYMBOLS XI CHAPTER INTRODUCTION 1.1 Part 1.1.1 Cell cycle in brief .2 1.1.2 DNA damage checkpoint (DDC) .7 1.1.3 DNA repair .14 1.1.4 Recovery and adaptation 17 1.1.5 Polo-like kinase, Cdc5 .20 1.2 Part II .22 1.2.1 Gene Expression 23 1.2.2 Oscillating Transcriptional Factors 23 1.2.3 Genome widespread oscillating transcription 24 1.2.4 Various examples of oscillating transcription 27 CHAPTER 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 Yeast strains and culture conditions 32 Plasmids .35 Yeast strains and culture conditions 35 Yeast transformation 36 Yeast DNA extraction 36 Southern blot analysis 37 Southern blot analysis with single-stranded probe 38 Real-Time PCR (RT-PCR) 40 Protein extraction using TCA 41 Western blot analysis .41 Protein extraction using acid washed glass beads 42 Co-immunoprecipitation 42 Sample preparation for SILAC mass spectrometry .43 Immunofluorescent staining (IF) .44 Microscopy 45 Flow cytometry analysis (FACS) .45 Effects of TF oscillations on gene expression .46 Numerical simulations .46 CHAPTER 3.1 3.2 MATERIALS AND METHODS 32 ADAPTATION IN CELLS WITH TELOPHASE TRAP .48 Background 48 Results 53 iii 3.2.1 The telophase trap using combined deficiencies of Cdc15 and Slk19.53 3.2.2 Recovery and adaptation in cells with telophase trap 54 3.2.3 Dramatic loss of viability in AD cells 58 3.2.4 Polo-like kinase Cdc5 is necessary for adaptation .61 3.2.5 Cdc5 polo kinase does not affect recovery in RP cells 64 3.3 Discussion 67 CHAPTER POLO-LIKE KINASE CDC5 AND ADAPTATION .71 4.1 Background 71 4.2 Results 74 4.2.1 Overexpression of Cdc5 accelerates adaptation 74 4.2.2 Overexpression of Cdc5 rescues other adaptation defective mutants 77 4.2.3 Resection of DNA at DSB is not affected by ectopic expression of Cdc5 84 4.2.4 High level of Cdc5 inhibits Ddc2 foci formation 87 4.2.5 Cdc5 inhibits formation of Ddc2 foci assembled at the site of DNA damage 93 4.2.6 Cdc5 polo kinase and the checkpoint clamp 96 4.2.7 A search for Cdc5 substrate(s) in adaptive response pathway .99 4.3 Discussion 103 CHAPTER REGULATION OF GENE EXPRESSION BY OSCILLATORY TRANSCRIPTION FACTORS 110 5.1 Background 110 5.2 Results 111 5.2.1 Formulation of gene expression models 111 5.2.2 Solutions of gene expression models .115 5.2.3 Estimation of trends .119 5.2.4 Oscillatory vs. non-oscillatory TF induction of gene expression 124 5.3 Discussion 130 CHAPTER 6.1 6.2 PERSPECTIVE AND FUTURE DIRECTIONS 135 Role of Cdc5 in Adaptation .135 Oscillating Transcription Factors .141 BIBLIOGRAPHY 145 APPENDICES 164 iv Summary Cells frequently incur genetic damage during their life times. To counter these, eukaryotic cells have evolved surveillance mechanisms, known as checkpoint controls, to detect such damages. When activated, the checkpoint pathways transiently halt progression through the division cycle. This allows cells to repair the DNA damage by either homologous recombination (HR) or non-homologous end joining (NHEJ). Once the DNA damage is repaired, the cell cycle can resume. Such corrective measures are critical to the maintenance of genomic stability through successive cell divisions. Cells defective in checkpoint controls accumulate chromosomal aberrations which may eventually lead to uncontrolled division or, in extreme cases, even cell death. Checkpoint pathways are frequently found to be defective in human tumors. However, in some instances when repair responses cannot be mounted, the cells escape the DNA damage imposed arrest and progress to mitosis with damaged DNA. This behavior, which is detrimental to chromosome stability, is known as adaptation. This phenomenon was first observed in the budding yeast Saccharomyces cerevisiae (the organism used in this study) [1-3], subsequently, adaptation was also found to occur in Xenopus [4], and in human cells [5]. Although Polo kinase (Cdc5 in yeast) is known to be required for adaptation [3], its exact role remains to be elucidated. In the first part of this study, we have investigated the role of Cdc5 polo kinase in the adaptive response. We have developed a new method to quantify adaptation in a cell population and have found that ectopic expression of Cdc5 accelerates the adaptation response. This is consistent with our observation that the level of Cdc5 v increases as cells prepare to undergo adaptation. High level of Cdc5 activity also suppresses the adaptation defect in cells deficient in Sae2, Ptc2, Ptc3 or Ckb1. We have shown that the requirement of Cdc5 for adaptation is not because of its role in mitotic progression as is generally believed. Instead, Cdc5 activity is necessary for extinguishing the checkpoint and to turn off the checkpoint by inhibiting the recruitment of checkpoint response protein Ddc2 to an unrepaired double-strandbreak (DSB). In addition, our model suggests that the prolonged period of G2/M arrest imposed before adaptation, could provide sufficient time for cells to accumulate enough Cdc5 activity needed to overcome the initial inhibition imposed by DNA damage-activated Rad53. Collectively, our results strongly suggest that Cdc5 polo kinase regulates upstream signaling events in the checkpoint pathway during the adaptive response. In the second part, a mathematical approach was taken to analyze the gene expression response due to oscillatory transcription factors (TFs). Oscillatory TFs have been reported in many diverse biological processes such as the: circadian clock [6, 7], somite segmentation during development [8], DNA damage response [9-12], inflammation [13-15], cell cycle [16, 17] and yeast glucose metabolism [18] (see Table 1). The resultant oscillatory gene expression appears to have specific functions. For instance, different oscillating dynamics of NF-B activates specific sets of genes [13], and p53 exhibits oscillatory profiles depending on the extent of DNA damage [9, 19, 20]. The effects elicited by oscillating TFs have been demonstrated, but are not well understood. Our findings stem from the estimated trends of gene expression responses we have modeled. It appears that the various effects caused by oscillatory TFs are intrinsic to the system. As an example (as shown in Figure 24), we have demonstrated vi a Hill kinetic system which involves oscillating gene expression and exhibits differential regulation of different target genes, suggesting that an oscillating TF could up-regulate a set of genes while others could be down-regulated or remain unchanged. These changes would not be possible for non-varying TFs without involving other entities. Therefore, oscillatory behaviour provides the TFs with additional degree of dynamics and.renders specificity to their responses. In the context of DNA damage in mammalian cells, an attractive possibility is that p53 may possess similar molecular properties that allow it to selectively up-regulate apoptotic genes upon severe DNA damage. Taken together, our analyses of the mathematical gene-expression models uncover a plausible mechanism to differentially regulate genes - an interesting intrinsic property hidden within the regulatory landscape relevant to gene expression. vii List of Tables Table 1. DNA damage checkpoint proteins .9 Table 2. Summary of reported oscillatory gene expressions in key biological processes. .25 Table 3. Summary of reported oscillations in protein synthesis 26 Table 4. Summary of reported oscillatory gene expressions induced under specific stimuli. .26 Table 5. Yeast strains used in this study. .34 Table 6. List of plasmids used in this study .35 Table 7. List of antibodies used in this study .41 Table 8. Cdc5 candidate substrates 102 Table 9. Possible Cdc5 phosphorylation and binding sites in Rfa1 .102 Table 10. Directionality of target protein trends as a function of Models M and H parameters 121 viii Appendices mp k k5 j1n Aa 1  j n Since wX p wPa w mp Xa Đ 4S ãĐ 4S ã ăă k  ááăă k5  áá Pa ạâ Pa â = (Xp is independent on T.A. oscillation period), wm p wAp and X p wPa wPa w Ap . Therefore, the effect of Pa on mp is qualitatively similar to the effect X p wPa2 wPa2 of Pa on Ap, i.e., wm p w 2mp > and wPa wPa2 < 0. j1n Aa .  j1n X a When Pa ặ 0, mp ặ 0; when Pa ặ f, mp ặ mm = mp j1n Aa  j1n X a Pa Effect of T.A. binding affinity to DNA enhancer site, j1 wmm wj1  2nk X an  ma2 >1  m  1  m @ư n n n a a k 42  wa2 w mm wj 12 j1n1 đ j1n 1  ma n  j1n 1  ma n  X an  ma2 >  @ > n n ẵ ắ!0  2nk X an  ma2 >1  m  1  m @ j n n n a > a n2 ^2n 1 X n a 1  m  j1n n  1  ma  1  ma n a  k  w j 1  ma  j 1  ma  X  m a n n When j1 = 0, mm = 0; as j1 ặ f, mm ặ n = 1: w mm < 0. As j1 ặ f, mm ặ wj 12 n n n a @ n n a @` ê 1  ma n  1  ma n . ô n nằ k 42  wa2 1  ma  1  ma ẳ k4 k4 k 42  wa2 mm ma . k4 k 42  wa2 ma j1 203 Appendices n = 2: When j1 = 0, w mm w mm ặ f , > 0. As j < 0, mm ặ wj 12 wj 12 > stationary point of inflexion occurs at j   ma2 1 m 2ma . A non2 k 42  wa2 ma  k4  1  m @ 2 a 2 a X n2 . mm 2m a k4 k 42  wa2 m a  j1  m a2 n = 4: When j1 = 0, 1  m  1  m 2 a 2 a w mm w mm ặ f , > 0. As j < 0, mm ặ wj 12 wj 12 Two non-stationary points of inflexion at ji = and j   ma2 1 m  1  m @ a a X n4 . mm 4m a m  k  w m a  6m a  a k4 >  4ma ma2  . k 42  wa2 ma  6ma  k4 a j1  m a2 1  m  1  m a w mm w mma > 0. As j < 0, m ặ f , ặ n = 8: When j1 = 0, m wj 12 wj 12  8ma ma6  ma4  7ma2  . Two non-stationary points of inflexion at ji = k 42  wa2 ma  28ma  70ma  28ma  mm 14  ma2 and j18 . X m m  m  m   k a a a a n 8  ma2   ma2 k  w m a  28m a  70m a  28m a  k4 >   @ a j1 14  m a2 1  m  1  m a a 204 Appendices Since 1  ma n  1  ma n ! 1  ma n1  1  ma n1 , m > m as j ặ f. m,n m,n-1 1  ma n  1  ma n 1  ma n1  1  ma n1 mm n=8 n=4 n=2 n=1 j1 Effect of stoichiometry of T.A. or binding cooperativity between T.F. and DNA enhancer site, n When n = 0, mm = 0. Note: mm wmm wn or wmm wn n n ê j1n  X a  Aa  j1n  X a  Aa ô ằ n n n k 42  wa2 ôơ j1n  X a  Aa  j1n  X a  Aa  2X a2  Aa2 ằẳ k4 k4 k 42  wa2 nê X 1  ma X 1  ma  ma n n  X an 1  ma ln a  X an 1  ma ln a 2k j1n 1  ma2 ô j1n ln ằ j1 j1  ma ẳ > k 42  wa2 j1n 1  ma  j1n 1  ma  X an 1  ma2 n @ n n nê  X  Aa   X  A n ln  X a  Aa X  Aa n 2k j1n X a2  Aa2 ô j1n ln a   X a  Aa ln a a a ằ X a  Aa j1 j1 ẳ  >  k 42  wa2 j1n  X a  Aa  j1n  X a  Aa  X a2  Aa2 n n @ n wmm j3 |n D ln 2 . How to use this info? wn X a  Aa Hence, 205 Appendices ê X 1  ma ê X a 1  ma wmm is always positive when ln ô a ằ d and ln ô ằ t , i.e., wn j1 j1 ẳ ẳ ơ Xa(1  ma) d j1 d Xa(1 + ma). wmm can be zero, positive or negative when j1 < Xa(1 ma) or j1 > Xa(1 + ma). wn Xa(1  ma) d j1 d Xa(1 + ma): k4 k w a wAm ! . Note: when j1 = Xa(1  ma), as n ặ f, mm ặ wn ; when Xa(1  ma) < j1 d Xa(1 + ma), as n ặ f, mm ặ k4 k  wa2 . Discuss the biological implication that mm as n ặ f depends on wa k4 k 42  wa2 mm Xa(1  ma) < j1 d Xa(1 + ma) k4 k 42  wa2 j1 < Xa(1 ma): wX M |n wn > 0. j1 = Xa(1  ma) n wmm can be zero, positive or negative. As n ặ f, mm ặ wn 0. Since mm is also zero at n = 0, mm cannot have a minimum value to satisfy the mm condition, mm t 0. n j1 > Xa(1 + ma): > k4 k 42  wa2 wmm can be zero, positive or negative. As n ặ f, mm ặ wn k4 k 42  wa2 . mm for all n. Hence, no maximum point. 206 Appendices mm k4 k  w a2 n Effect of T.A. oscillation amplitude, ATF wmm wATF w mm wATF 2nk j1n k 42  wa2 X a  Aa2 >j X n >2 j n 1 X a   X a  Aa n 1  n n  n @  > >  2nk j1n X a2  Aa2 n ^ @ @! @ @  n > @ ê j12 n X a X a2  Aa2  X a  Aa n1   X a  Aa n1 ẵ ằ ô n ằ ô 12 j1n Aa X a X a2  Aa2 ằ  2n ô ằắ 2n 2n 2 n ằ ằ ô j1 X a  Aa  X a  Aa   X a  Aa ằẳ ằ ô n 1 n 1 2 n ằẳ ôơ Aa X a  Aa  X a  Aa   X a  Aa @ằ    > > @ @ n2  k 42  wa2 j1n  X a  Aa  j1n  X a  Aa  X a2  Aa2   2 n a n ê 4(n  1) j1n Aa X a j1n  X a  Aa n  j1n  X a  Aa n  X a2  Aa2 ô n 1 n n đô  X a  Aa >(n  1) X a  (3n  1) Aa @ j1  2 X a  Aa ô n 1 n n ôơ  X a  Aa >(n  1) X a  (3n  1) Aa @ j1  2 X a  Aa > n 1 n2 k 42  wa2 j1n  X a  Aa  j1n  X a  Aa  X a2  Aa2 >   X a  Aa  Aa  j1n  X a  Aa  X a2  A n a 2nk j1n X a2  Aa2 > n n > @ n @ > ư X  A n n  j n A X   X  A n    X  A n   j n 3n  X  n  A a a a a a a a a a a n n 2n đ j1 X a  Aa  nX a  X a  Aa   Aa  nX a  X a  Aa n 2n2 2n2  j n   X a  Aa   X a  Aa > > When Aa = 0, mm = 0, @ @ w mm > 0. As Aa ặ Xa, mm ặ wATF w mm n = 1: > 0. wma2 k4 k  wa2 @`ẵ ắ . mm k4 k 42  wa2 Aa w mm < as Aa ặ Xa. For n = 2, wATF Xa 207 Appendices w mm = as Aa ặ Xa. wATF For n > 2, mm k4 k 42  wa2 n>2 n=2 Aa Effect of T.A. oscillation mean, Xa wmm wX a  w mm wX a2 X 2nk j1n k 42  wa2 > a  Aa2 >j X n >2 j n1 n Aa   X a  Aa n1  n  n2 a 2nk j1n X a2  A 2 n a  k 42  wa2 j1n  X a  Aa  j1n  X a  Aa  X a2  Aa2  n n > ư 2n  X  A2 n j n A X   X  A n2   X  A n2 a a a a a a a a n n 2n đ j1 Aa  X a  nAa  X a  Aa   X a  nAa  X a  Aa n n2 n   X a  Aa  j1 n   X a  Aa > > As Xa ặ Aa, mm ặ n=1: w mm > 0. wX a2 @  Aa  j1n  X a  Aa  X a2  A n a n 1 XaX a  Aa @ @ k4 k42  wa2 ; as Xa ặ f, mm ặ 0, k4 @ @ n @ẵ ắ w mm ặ 0. wX a2 mm k 42  wa2 Xa Aa For n = 2, w mm < as As Xa ặ Aa. Thus, a point of inflexion must exist. wX a2 mm k4 k  wa2 Xa Aa 208 Appendices For n > 2, w mm = as As Xa ặ Aa. Thus, a point of inflexion must exist. wX a2 mm k4 k  wa2 Xa Aa Effect of rates of transcription (k1) and translation (k2) Both mm and mp are independent of k1 and k2. mm, mp k1, k2 Effect of degradation rates of mRNA (k4) and protein (k5) wmm wk w mm wk 42 1  j n j1n Aa wa2  Xa k  w  1  j n a !0 a j Aa w k n  X a k 42  wa2 0 j1n Aa . (discuss that it is very interesting that 1 j1n X a a high degradation rate promotes mm but not vice-versa as anticipated!!!) When k4 = 0, mm = 0; as k4 ặ f, mm ặ mm j1n Aa  j1n X a k4 209 Appendices wmm wk wm p wk w 2mp wk 52 0. k mm wa2   wa2 !0 3mm wa2 k k  wa2 0 When k5 = 0, mp = 0; as k5 ặ f, mp ặ mm. (discuss that it is very interesting that a high degradation rate promotes mp but not vice-versa as anticipated!!!) mp mm k5 D. Oscillation period of target gene wm wa Pm Pa wp wa Pp Pa The oscillation periods of the target gene mRNA and protein level were compared with the oscillatory T.A. period. In agreement with the analytical solution, the oscillation periods of the target gene mRNA and protein levels are identical to the oscillation period of the T.A. for all simulation runs. E. Time-delay of target gene T m W1  Đw ã arctanăă a áá wa â k4 T p W1 W  wa ê Đ wa ã Đw ã Đ wa ã arctanăă a áá ôarctanăă áá  arctanăă ááằ T m  W  wa â k4 â k ạẳ â k5 210 Appendices Effect of transcription activator oscillation frequency (wa) wT m wwa w 2T m wwa2 Note: ê Đw ã ô ăă a áá ằ Đ wa ãằ ô â k4  arctanăă ááằ  wa2 ô Đ w ã â k ạằ ô1  ă a ôơ ăâ k áạ ằẳ ê Đ wa ã ăă áá ằ ô 2 Đ wa ã ô â k ằ  k k  3wa ă arctan  2ằ ăk wa3 ô Đ wa ã ằ wa2 k 42  wa2 â 4ạ ô  ăă áá ôơ â k ằẳ   wT m w 2T m f x  > @ < as  arctan f x for f(x) > 0. can be positive, zero or wwa wwa2  > f x @ negative. As wa ặ 0, Tm ặ W  . As wa ặ f, Tm ặ W1. (discuss that the resultant time-delay is k4 always longer than the transcriptional time-delay by 1/k4, i.e., the self-degradation rate of mRNA influences its time-delay!!!) Thus, W1  k4 Tm W1  w 2T m !0 wwa2 W1 k4 Tm w 2T m increases wwa2 from ve to +ve W1 wa wa Similarly, 211 Appendices wT p wwa w 2T p wwa2 ưê Đ w ã ê Đw ã ẵ ô ă a ằ ô ăă a áá ằ ă Đ wa ãằ ô â k Đ wa ãằ ô â k ă ă   arctan arctan  đ ă k áằ ắ  ă k áằ ô wa2 ô Đ w ã â ạằ â ạằ ô Đ wa ã ô1  ă a 1 ă ô ă k ằ ô ăâ k áạ ằẳ ẳ ơ â Note: w 2T p wwa2 can ưê Đ wa ã ê Đ wa ã ẵ ô ăă áá ằ ô ăă áá ằ 2 k k Đ wa ã Đ ã w k k  3wa2 ô ằ ô ằ k k  3wa â a  ô2 arctanăă áá  â ằ ắ  5  4 đ arctanăă áá  2ằ 2 2 k k wa3 ô wa2 k 42  wa2 Đ wa ã ằ wa k  wa Đ wa ã ằ ô â 4ạ â 5ạ ô ă ă  1  ô ă k ằ ăk ằ ô â ẳ â 5ạ ẳ ơ     be positive, zero or negative. Đ1 1ã As wa ặ 0, Tp ặ W  W  ăă  áá . As wa ặ f, Tp ặ W1 + W2. (discuss that the â k k5 resultant time-delay is always longer than the transcriptional and translational time-delay by 1/k4 & 1/k5, i.e., the self-degradation rate of mRNA & protein influences its timedelay!!!) Thus, Tp 1  k k5 Tp w 2T p ww a 1  k k5 !0 W1  W w 2T p increases wwa2 from ve to +ve W1  W wa Effect of degradation rates of transcriptional time-delay (W1) and translational time-delay (W2) Tm, Tp Tm W1 Tp W2 Effect of degradation rates of mRNA (k4) 212 Appendices wT m wk  w 2T m wk 42 0 k  wa2 k 2k 4  wa2 !0 As k4 ặ 0, Tm ặ W  S wa . As k4 ặ f, Tm ặ W1. (similar to the effect of wa, the resultant time-delay is always longer than the transcriptional and translational time-delay by 1/wa) W1  Tm S wa Similarly, wT p  wk w 2T p k wk 42 W1 2k 4  wa2 k4 !0 ặ 0, Tp ặ k4 As 0 k  wa2 W1 W  W1 W  wa êS Đ wa ã ô  arctanăă ááằ . â k ạẳ ơ2 As k4 ặ f, Tp ặ Đw ã arctanăă a áá . (similar to the effect of wa, the resultant time-delay is always wa â k5 longer than the transcriptional and translational time-delay by 1/wa) Tp S wa W1  W  Đw ã arctan ăă a áá wa â k5 k4 Effect of degradation rate of protein (k5) wT p wk w 2T p wk  0 k  wa2 k 2k 5  wa2 !0 213 Appendices As k5 W1 W  ặ 0, Tp ặ W1 W  wa êS Đ wa ã ô  arctanăă ááằ . â k ạẳ ơ2 As k5 ặ f, Tp ặ Đw ã arctanăă a áá . (similar to the effect of wa, the resultant time-delay is always wa â k4 longer than the transcriptional and translational time-delay by 1/wa) S wa Đw ã W1 W  arctan ăă a áá wa â k4 Tm Tp k5 214 Appendices 7.5 Simulation source codes Dynamics_A1_constant_validate.m.txt k1 = 0.1*1000*60; % [0.001, 1]*1000*60 nM/hr = {0.001, 0.01, 0.1, 1}*1000*60 j1 = 0.53*1000; % [0.01, 10]*1000 nM = {0.01, 0.1, 1, 10}*1000 --> k2 = 0.01*60; % [0.001, 1]*60 /hr = {0.001, 0.01, 0.1, 1}*60 dM = 0.01*60; % [0.001, 1]*60 /hr = {0.001, 0.01, 0.1, 1}*60 -->; large effect on time to steady state (large dM => fast; small dM => slow) dP = 0.01*60; % [0.001, 1]*60 /hr = {0.001, 0.01, 0.1, 1}*60 n = 4; % {1, 2, 4, 8} ; large effect on time to steady state (large dM => fast; small dM => slow) dP = 0.01*60; % [0.001, 1]*60 /hr = {0.001, 0.01, 0.1, 1}*60 n = 1; % {1, 2, 4, 8} ; large effect on time to steady state (large dM => fast; small dM => slow) dP = 0.01*60; % [0.001, 1]*60 /hr = {0.001, 0.01, 0.1, 1}*60 n = 4; % {1, 2, 4, 8} ; large effect on time to steady state (large dM => fast; small dM => slow) dP = 0.01*60; % [0.001, 1]*60 /hr = {0.001, 0.01, 0.1, 1}*60 n = 1; % {1, 2, 4, 8} [...]... protect the genome integrity One such mechanism, the DNA damage checkpoint, inhibits cell cycle progression in response to DNA damage and causes cells to arrest in G2/M until the offending lesions are repaired A failure to repair DNA damage prolongs the G2/M arrest However, cells eventually escape the checkpoint arrest and progress through mitosis with damaged chromosomes [1-3] This cellular behavior has... replication checkpoint, DNA damage checkpoint (DDC) and spindle assembly checkpoint (SAC) The morphogenetic checkpoint monitors conditions that affect proper bud formation [59] It responds to a defect in bud emergence and delays the initiation of nuclear division The replication checkpoint is triggered if DNA synthesis is disrupted, while the DDC responds to any insult to the DNA such as modification of nitrogenous... full activation by trans-autophosphorylation The activated kinases Rad53 and Chk1 are together responsible for a number of downstream responses These include DNA damage- induced transcriptional regulation, increase in the dNTP pool for DNA repair and inhibition of the progression to mitosis [81] 11 Chapter 1 Introduction Figure 1 Proteins involved in DNA damage responses due to a DSB (a) MRX complex (Mre11,... Plk1-mediated degradation of Wee1 is essential for recovery from a DNA damage- induced arrest but not for mitotic entry during a normal cell cycle [112] Phosphorylation of Wee1 by Plk1 leads to Wee1 degradation, and also to less inhibition on Cdk1/cyclin B complex [112] Adaptation to DNA damage checkpoint has also been reported recently in human cells [5] Following ionizing radiation -induced damage, human osteosarcoma... taken up into the presynaptic filament to base pair with the initiating ssDNA, resulting in the extension of the initial DNA joint This process is termed "DNA strand exchange" or "DNA branch migration" The extent of DNA branch migration is determined by the length of the presynaptic filament The homologous DNA is used as a template to synthesize new DNA, and forms a D loop structure [95] The DNA structures... loaded onto the RPA coated ssDNA by Rad24– RFC clamp loader [69] In the absence of RPA, 9-1-1 can be loaded onto DNA with either a 3’- or 5’-junction [70] However, if the DNA is coated with RPA, loading of 9-1-1 complex onto the 3’-junction of the DNA is prevented As for Mec1-Ddc2, its binding to RPA appears to be intrinsic in that the recruitment of Mec1-Ddc2 complex depends on a conserved checkpoint. .. timing of S phase initiation and, in late telophase when it participates in the inactivation of mitotic kinase to aid the final exit from mitosis 1.1.1.4 Cell cycle regulation by checkpoint pathways To ensure proper functioning of the cell cycle, the cells employ checkpoints that monitor various cellular events [58] Four major checkpoints are now known in the budding yeast: morphogenetic checkpoint, DNA. .. cycle The onset of M phase (also referred to as simply ‘mitosis’) is catalyzed by a kinase complex formed by association of Cdc28 with the mitotic cyclins (Clb1, Clb2, Clb3, Clb4) Cdc28-Clb2 kinase contributes a major part of the total Cdc28 mitotic activity [29-31] At the time of entry into mitosis, the inter-SPB bridge is broken and cells assemble a mitotic spindle In addition, kinetochore-microtubules... involvements during mitosis, cdc5ǻ mutant cells can still transit through metaphase and anaphase, but are unable to exit mitosis eventually arresting with a large bud, a long spindle and a divided nucleus This suggests that the regulation of mitotic exit may be the sole essential function of Cdc5 kinase in mitotic cycle of budding yeast Besides mitosis, Cdc5 is also implicated in DNA damage checkpoint and... homologous DNA pairing and strand exchange In response to DNA damage and subsequent generation of ssDNA, the RAD52 epistasis group mediates replacement of RPA with Rad51 [94] Like RecA, Rad51 also forms a right-handed helical filament on ssDNA in which the DNA is held in an extended state The Rad51-ssDNA nucleoprotein filament (also known as the presynaptic filament) contains a binding site for dsDNA and . ANALYSIS OF ADAPTIVE RESPONSE TO DNA DAMAGE CHECKPOINT INDUCED ARREST YIO WEE KHENG NATIONAL UNIVERSITY OF SINGAPORE 2009 ANALYSIS OF ADAPTIVE RESPONSE TO DNA DAMAGE CHECKPOINT INDUCED. the DNA damage checkpoint, inhibits cell cycle progression in response to DNA damage and causes cells to arrest in G2/M until the offending lesions are repaired. A failure to repair DNA damage. discussion of DNA damage checkpoint control and events associated with it, it is useful to begin with a brief description of the general regulatory landscape of the cell cycle in which the DNA damage